PROJECT SUMMARY/ABSTRACT A major challenge in ending the HIV pandemic is the persistence of the latent HIV reservoir that can lead to viremia, disease progression and transmission to new hosts after discontinuation of antiretroviral therapy (ART). Efficient treatment regimens that successfully eliminate cell populations carrying intact proviruses are not available due to the incomplete understanding of the cellular mechanisms that allow the virus to remain quiescent within the host genome. A compounding risk factor in HIV pathology is substance use disorder (SUD) known to contribute to decreased adherence to and delay of viral decay due to ART, and accelerated progression to AIDS. We hypothesize that persistence of HIV reservoirs is encoded in the proviral location within the 3D architecture of the host genome (nucleome), and influenced by SUD-induced changes in epigenetic structures. During the exploratory high-risk R61 phase of this project, we will first identify SUD-related differences in HIV integration site patterns, the epigenome and the nucleome of CD4+ T lymphocytes derived from patient cohorts that differ in their exposure to methamphetamine (meth). In the second aim, we will apply advanced 3D nucleome mapping strategies to define the interdependence of nuclear structure, proviral genomic location, regulatory elements, and transcriptomes in short-term ex vivo cultivated patient-derived cells from both cohorts. In the third aim, we will use gene-editing techniques to precisely define a role for proviral integration sites (identified in aims 1 and 2) in cellular gene activity and HIV persistence, including those related to meth use. In the R33 phase we propose to assess the impact of targeted provirus integration on nucleome architecture and transcriptomes to clearly identify genes essential for establishing the ART-resistant HIV reservoir. In addition, we will pursue a strategy to define chromatin interactions of proviral integrations and host genes in vivo in human CD4+ T cell populations of HIV-infected individuals.